Compensated patch-clamp amplifier for nanopore polynucleotide sequencing and other applications

Abstract

A compensated patch-clamp system for polynucleotide sequencing and other applications.

1 . A patch-clamp system, comprising:
 a circuit producing timing signals;   a differential amplifier circuit having a non-inverting input, an inverting input with a parasitic capacitance and connected to an electrode resistance, and an output;   a feedback resistor connected between said output and said inverting input;   a reset switch receiving said timing signals, said reset switch for selectively connecting said output to said inverting input in response to said timing signals;   a command voltage circuit receiving timing signals and command voltages, said command voltage circuit for applying stepped command voltages to said non-inverting input in response to said timing signals; and   a sensor having an input capacitance and a series resistance, said sensor operatively connected to said inverting input;   wherein said reset switch is closed in synchronization with a step change in said stepped command voltages for a time TR;   wherein said reset switch is opened after said time TR;   wherein said time TR is sufficient to prevent saturation of said differential amplifier circuit without blanking said stepped voltage; and   wherein said stepped command voltages are selected to compensated for said series resistance and said electrode resistance to produce a predetermined voltage across said sensor.   
     
     
         2 . The patch-clamp system according to  claim 1 , wherein said sensor comprises a nanopore sensor. 
     
     
         3 . The patch-clamp system according to  claim 1 , wherein said differential amplifier circuit includes a current-to-voltage converter and a difference amplifier. 
     
     
         4 . The patch-clamp system according to  claim 1 , wherein said command voltage circuit comprises a sample and hold circuit. 
     
     
         5 . The patch-clamp system according to  claim 1 , wherein said command voltage circuit comprises a digital-to-analog converter. 
     
     
         6 . The patch-clamp system according to  claim 5 , wherein said output is applied to an analog-to-digital converter that produces an amplified digital version of said current in said sensor. 
     
     
         7 . The patch-clamp system according to  claim 6 , wherein said amplified digital version is applied to a field programmable array. 
     
     
         8 . The patch-clamp system according to  claim 6 , wherein said amplified digital version is input to a computer. 
     
     
         9 . The patch-clamp system according to  claim 8 , wherein said computer causes said command voltages to be applied to said command voltage circuit. 
     
     
         10 . A method of compensating a sensor in a patch-clamp system, comprising the steps of:
 a) connecting a first end of an electrode to an inverting input of a patch-clamp system;   b) connecting the second end of the electrode to ground;   c) connecting a feedback resistor RF between the inverting input and the output of the patch-clamp system;   d) obtaining a steady state output of the patch-clamp system by setting the voltage on the non-inverting input to a reference voltage;   e) applying a step voltage to the non-inverting input;   f) determining the output voltage variation of the patch-clamp system converter in response to the step voltage;   g) calculating the electrode series resistance RE using the output voltage variation determined in step f);   h) connecting a sensor between the second end of the electrode and ground;   i) obtaining a steady state output of the patch-clamp system by setting the non-inverting input to a reference voltage;   j) measuring the sensor current i after the steady state output is achieved;   k) determining the sensor series resistance RS from the measured sensor current i, the electrode series resistance RE, and the steady state output;   l) using the sensor by obtaining a predetermined voltage across the sensor by applying a compensated voltage to the non-inverting input where the compensated voltage is equal to the predetermined voltage plus the sensor current i times the sensor series resistance RS.   
     
     
         11 . The method of compensating a sensor in a patch-clamp system according to  claim 10 , further including the steps:
 activating the patch-clamp system to achieve a steady state response after the sensor series resistance RS has been determined;   applying a compensation step voltage to a non-inverting input of the patch-clamp system;   determining the time constant of the output of the patch-clamp system to the compensation step voltage; and   determining the input parasitic capacitance of the sensor from the sensor series resistance RS and the determined time constant.   
     
     
         12 . The method of compensating a sensor in a patch-clamp system according to  claim 11 , further including the step of determining a reset pulse width based on the determined input parasitic capacitance. 
     
     
         13 . A nanopore sequencer, comprising:
 a nanopore sensor having an input resistance RN and an input capacitance CN;   a patch-clamp circuit having a non-inverting input, an inverting input with a parasitic capacitance CP, and an output;   an electrode connecting said nanopore sensor to said inverting input, said electrode having an electrode series resistance RE;   a feedback resistor connected between said output and said inverting input;   a reset switch receiving timing signals, said reset switch for selectively connecting said output to said inverting input in response to said timing signals;   a digital-to-analog circuit receiving timed digital command voltages, said digital-to-analog circuit for applying stepped command voltages to said non-inverting input in response to said timed digital command voltages; and   wherein said reset switch is closed in synchronization with a step change in said stepped command voltages for a time TR;   wherein said reset switch is opened after said time TR;   wherein said time TR is sufficient to prevent saturation of said patch-clamp circuit without blanking said stepped voltage; and   wherein said stepped command voltages are selected to compensate for said input resistance RN and said electrode series resistance RE so as to produce a predetermined voltage across said nanopore sensor.   
     
     
         14 . The nanopore sequencer according to  claim 13 , wherein said nanopore sensor comprises a semi-conductive material. 
     
     
         15 . The nanopore sequencer according to  claim 13 , wherein said nanopore sensor comprises a cell membrane. 
     
     
         16 . The nanopore sequencer according to  claim 13 , wherein said  1 , wherein said patch-clamp circuit includes a current-to-voltage converter and a difference amplifier. 
     
     
         17 . The nanopore sequencer according to  claim 13 , wherein said output is applied to an Analog-to-Digital converter that produces an amplified digital version of said current in said nanopore sensor. 
     
     
         18 . The nanopore sequencer according to  claim 17 , wherein, wherein said amplified digital version is input to a field programmable array. 
     
     
         19 . The nanopore sequencer according to  claim 17 , wherein said amplified digital version is input to a computer. 
     
     
         20 . The nanopore sequencer according to  claim 13 , wherein said computer operatively produces said timing signals and said timed digital command voltages. 
     
     
         21 . The nanopore sequencer according to  claim 13  adapted to sequence a polynucleotide.